Tyrosine kinase

ABSTRACT

Disclosed are a novel cytoplasmic tyrosine kinase which is increased with respect to expression amount thereof in accordance with the differentiation of blood cells, and a deoxyribonucleic acid (DNA) coding for the same. The tyrosine kinase of the present invention can be advantageously used for screening chemical substances having the capability to inhibit or activate the tyrosine kinase activity of at least the tyrosine kinase of the present invention. Also disclosed are a replicable recombinant DNA molecule comprising a replicable expression vector and, operably inserted in the vector, a DNA coding for the tyrosine kinase of the present invention; a microorganism or animal cells transformed with the replicable recombinant DNA molecule; an antibody reactive with a polypeptide comprising as an immunogen at least part of an amino acid sequence of the tyrosine kinase of the present invention; a sense DNA prepared from the cDNA coding for the tyrosine kinase of the present invention and an anti-sense DNA which is complementary to the sense DNA; and a sense RNA prepared from the cDNA coding for the tyrosine kinase of the present invention and an anti-sense RNA which is complementary to the sense RNA.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel tyrosine kinase and adeoxyribonucleic acid (DNA) coding for the same. More particularly, thepresent invention is concerned with a novel cytoplasmic tyrosine kinasewhich is increased with respect to expression amount thereof inaccordance with the differentiation of blood cells, and is alsoconcerned with a DNA coding for the same. The tyrosine kinase of thepresent invention can be advantageously used for screening chemicalsubstances having the capability to inhibit or activate the tyrosinekinase activity of at least the tyrosine kinase of the presentinvention. Therefore, the present invention is also concerned with amethod of screening for such chemical substances. The present inventionis also concerned with a replicable recombinant DNA molecule comprisinga replicable expression vector and, operably inserted in the vector, aDNA coding for the tyrosine kinase of the present invention; amicroorganism or animal cell transformed with the replicable recombinantDNA molecule; an antibody reactive with a polypeptide comprising as animmunogen at least part of an amino acid sequence of the tyrosine kinaseof the present invention; a sense DNA prepared from the cDNA coding forthe tyrosine kinase of the present invention, and an anti-sense DNAwhich is complementary to the sense DNA; and a sense RNA prepared fromthe cDNA coding for the tyrosine kinase of the present invention and ananti-sense RNA which is complementary to the sense RNA.

2. Discussion Of Related Art

Human blood contains various types of blood cells, and each of themplays a physiologically important role. For example, erythrocytes carryoxygen in a human body, platelets block bleeding, and leukocytes form animmune system to protect a human body against is infection. Thesevarious types of blood cells are derived from hematopoietic stem cellsin bone marrow.

Recent studies show that hematopoietic stem cells undergodifferentiation into various types of blood cells, osteoclasts, mastcells and the like, when stimulated by various hematopoietic-stimulatingfactors or various environmental factors. However, the mechanism of thedifferentiation of hematopoietic stem cells has not yet been fullyelucidated. Recent studies also show that a tyrosine kinase greatlyparticipates in the development and differentiation of bodies of animalsand insects. It has been considered that a tyrosine kinase does alsogreatly participate in the differentiation of hematopoietic stem cells.For example, it has been reported that c-kit, which is one of receptortyrosine kinases, is expressed on the surfaces of hematopoietic stemcells, and functions as a receptor for a hematopoietic growth factor anda mast cell growth factor (see Witte, Cell 63: 5, 1990). This receptortyrosine kinase controls the differentiation of hematopoietic stemcells.

Tyrosine kinases are enzymes which phosphorylate tyrosine residues of aprotein, and the physiologically active sites thereof each consist ofabout 250 amino acid residues. Tyrosine kinases each have a plurality ofwell conserved amino acid sequences (see Hanks et al., Science 241: 42,1988). A tyrosine kinase gene fragment may be obtained by preparing aDNA corresponding to the conserved amino acid sequence and conducting areverse transcription-polymerase chain reaction (RT-PCR) using theprepared DNA as a primer (see Wilks, Methods in Enzymology 200: 533,1991).

SUMMARY OF THE INVENTION

It is known that a tyrosine kinase plays an important role in control oftranscription and in signal transmission, and that a mutation of thegene coding for a tyrosine kinase, or a viral infection may causemalignant alteration of cells.

Tyrosine kinases are classified into two classes, i.e., those of thereceptor type and those of the cytoplasmic type. A cytoplasmic tyrosinekinase has not yet been elucidated as compared to a receptor tyrosinekinase, so that it has been strongly desired to make clear thecharacteristics of the cytoplasmic tyrosine kinase.

It is an object of the present invention to provide a novel cytoplasmictyrosine kinase which is considered to control the differentiation ofblood cells.

The present inventor has made extensive and intensive studies with aview toward developing a novel cytoplasmic tyrosine kinase. Morespecifically, the present inventor has conducted a cloning of the genecoding for a tyrosine kinase which participates in the differentiationof human megakaryoblastic leukemia cell line UT-7 (see Komatsu et al,.Cancer Res. 51: 341, 1991) (UT-7 is available from Dr. Norio Komatsu,instructor at the Department of Hematology, Jichi Medical School,Japan), utilizing the RT-PCR method. As a result, the present inventorhas unexpectedly found a gene fragment of a novel tyrosine kinase, themRNA of which is almost not expressed in undifferentiated UT-7, but isincreased with respect to expression amount thereof in accordance withthe differentiation of UT-7. The present inventor has used this genefragment as a probe to obtain a cDNA coding for the entire tyrosinekinase from the cDNA library of UT-7, and determined the nucleotidesequence of the obtained cDNA. Further, from the cDNA obtained, thepresent inventor has prepared anti-sense DNA and RNA, cells transformedwith the cDNA, and antibodies reactive with the tyrosine kinase, andconducted screening of chemical substances. Thus, the present inventionhas been completed.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

In each of SEQ ID NOs. 1 to 5, the left end and right end of the aminoacid sequence are the N-terminus and C-terminus, respectively. In eachof SEQ ID NOs. 6 to 11, the left end and right end of the nucleotidesequence are the 5'-end and 3'-end, respectively.

SEQ ID NO. 1 is the amino acid sequence of an SH3 domain (which isexplained below), which corresponds to the 7th to 70th amino acids inSEQ ID NO. 4 and the 48th to 111th amino acids in SEQ ID NO. 5, whereinboth SEQ ID NOs. 4 and 5 are the amino acid sequences of the tyrosinekinase of the present invention.

SEQ ID NO. 2 is the amino acid sequence of an SH2 domain (which isexplained below), which corresponds to the 81st to 155th amino acids inSEQ ID NO. 4 and the 122nd to 196th amino acids in SEQ ID NO. 5, whereinboth SEQ ID NOs. 4 and 5 are the amino acid sequences of the tyrosinekinase of the present invention.

SEQ ID NO. 3 is the amino acid sequence of a tyrosine kinase domain(which is explained below), which corresponds to the 192nd to 437thamino acids in SEQ ID NO. 4 and the 233rd to 478th amino acids in SEQ IDNO. 5, wherein both SEQ ID NOs. 4 and 5 are the amino acid sequences ofthe tyrosine kinase of the present invention.

SEQ ID NO. 4 is the amino acid sequence of one region which contains allof the SH3 domain, the SH2 domain and the tyrosine kinase domain. Theamino acid sequence of SEQ ID NO. 4 is coded for by the translationalregion started from the initiation codon at the 331st to 333rdnucleotides of the nucleotide sequence of SEQ ID NO. 11.

SEQ ID NO. 5 is the amino acid sequence of another region which containsall of the SH3 domain, the SH2 domain and the tyrosine kinase domain.The amino acid sequence of SEQ ID NO. 5 is coded for by thetranslational region started from the initiation codon at the 208th to210th nucletoides of the nucletoide sequence of SEQ ID NO. 11, andcontains the amino acid sequence of SEQ ID NO. 4.

SEQ ID NOs. 6 to 10 are the respective nucleotide sequences of fragmentsfrom the nucleotide sequence of SEQ ID NO. 11. The nucleotide sequencesof SEQ ID NOs. 6 to 10 are examples of nucleotide sequences coding forthe amino acid sequences of SEQ ID NOs. 1 to 5, respectively.

SEQ ID NO. 11 is the entire nucleotide sequence of the cDNA coding forthe novel tyrosine kinase of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, there is provided an isolatedpolypeptide having tyrosine kinase activity, comprising an amino acidsequence selected from the group consisting of SEQ ID NOs. 1, 2, 3, 4and 5. Also, there is provided an isolated deoxyribonucleic acid codingfor the polypeptide.

In another aspect of the present invention, there is provided areplicable recombinant DNA molecule comprising a replicable expressionvector and, operably inserted in the vector, a deoxyribonucleic acidcoding for a polypeptide having tyrosine kinase activity, thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1, 2, 3, 4 and 5. Also, there is provided amicroorganism or animal cells transformed with the replicablerecombinant DNA molecule.

In a further aspect of the present invention, there is provided a methodof screening for chemical substances having the capability to inhibit oractivate tyrosine kinase activity, which comprises:

contacting a sample material with a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs. 1, 2, 3, 4and 5 to detect a chemical substance having the capability to inhibit oractivate the tyrosine kinase activity of at least the polypeptide,wherein the capability of the chemical substances is utilized as acriterion for the detection; and

isolating the detected chemical substance from the sample material.

In still a further aspect of the present invention, there is provided anantibody reactive with a polypeptide having tyrosine kinase activity,the polypeptide comprising as an immunogen at least part of an aminoacid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3,4 and 5.

In still a further aspect of the present invention, there is provided anisolated DNA fragment selected from the group consisting of a sense DNAcomprising at least 12 contiguous deoxyribonucleotides of thedeoxyribonucleotide sequence of SEQ ID NO. 11, and an anti-sense DNAwhich is complementary to the sense DNA. Also, there is provided anisolated RNA fragment selected from the group consisting of ananti-sense RNA comprising at least 12 contiguous ribonucleotides of aribonucleotide sequence complementary to the deoxyribonucleotidesequence of SEQ ID NO. 11, and a sense RNA which is complementary to theanti-sense RNA.

In the present invention, various the term "tyrosine kinase activity"means not only the enzyme activity to phosphorylate a tyrosine residue,but also the activity of the SH2 domain of the tyrosine kinase torecognize a phosphorylated tyrosine residue of other proteins and bindto the residue, and the activity of the SH3 domain of the tyrosinekinase to recognize the amino acid sequence of a proline-rich region ofother proteins and bind to the region.

In the present invention, various procedures, such as preparation of acDNA which is required for cloning, evaluation of the expression of anRNA by northern blotting, screening by hybridization, preparation of arecombinant DNA molecule, determination of a nucleotide sequence of aDNA, and preparation of a cDNA library, can be conducted according tothe methods described in standard laboratory manuals. With respect tothe illustrative method, reference can be made, for example, toMolecular Cloning, A laboratory manual, edited by Maniatis (1989, Eds.,Sambrook, J., Fritsch, E. F., and Maniatis, T., Cold Spring HarborLaboratory Press).

Preparation of a PCR primer corresponding to the amino acid sequencewhich is characteristic of tyrosine kinase, and subsequent operation ofPCR can be conducted according to the method described in the literatureby Wilks (Proc. Natl. Aced. Sci. USA 86: 1603, 1989). Illustrativelystated, an oligonucleotide is synthesized by means of a commerciallyavailable DNA synthesizer. The synthesized oligonucleotide is purifiedand then, subjected to PCR, so that the specific region coding for thekinase domain of the tyrosine kinase and having about 210 bp isamplified. The resultant DNA fragment obtained by PCR is isolated, forexample, by agarose gel electrophoresis, and the isolated DNA fragmentis purified. The purified DNA fragment is subcloned into variousvectors, followed by determination of the nucleotide sequence thereof.From the comparison of the determined nucleotide sequence with thenucleotide sequences of the various types of known tyrosine kinases,cloning of the nucleotide fragment of a novel tyrosine kinase can beconfirmed.

In order to obtain a clone containing the entire nucleotide sequencecoding for the novel tyrosine kinase of the present invention, thenucleotide fragment cloned by the above procedure is isotopically ornon-isotopically labeled and, a cDNA library of UT-7 is screened withthe labeled nucleotide fragment, for example, by hybridization. Examplesof methods for labeling a nucleotide fragment with an isotope include amethod in which the terminus of the nucleotide fragment is labeled with³² P!γ-ATP using T4 polynucleotide kinase, a nick translation method,and a primer extension method. Alternatively, a clone containing theentire nucleotide sequence coding for the novel tyrosine kinase of thepresent invention may be obtained by the PCR method, based on thenucleotide sequence of SEQ ID NO. 11.

SEQ ID NO. 11 is a cDNA nucleotide sequence coding for the tyrosinekinase of the present invention. The nucleotide sequence of SEQ ID NO.11 consists of the 5' non-coding region of 207 nucleotides, the regionof 1521 nucleotides coding for the novel tyrosine kinase, and the 3'non-coding region of 214 nucleotides.

SEQ ID NOs. 4 and 5 are the amino acid sequences of the tyrosine kinaseof the present invention. Each of the polypeptides of SEQ ID NOs. 4 and5 contains an SH2 domain, an SH3 domain and a tyrosine kinase domain,which respectively have similar amino acid sequences to the partialamino acid sequences of known cytoplasmic tyrosine kinases these threetypes of domains are known to be present in many types of knowncytoplasmic tyrosine kinases, e.g., c-src (see Koch et al., Science 252:668, 1991)!. Further, it has been reported that the SH2 domain and theSH3 domain are also present in signal transmitters, such asphospholipase C-γ, IP3 kinase, and ras-GAP, and that these two types ofSH domains play important roles in intracellular signaling pathways (seePawson and Gish, Cell, 71: 359, 1992). In the tyrosine kinase of thepresent invention, the SH3 domain has the amino acid sequence of SEQ IDNO. 1, the SH2 domain has the amino acid sequence of SEQ ID NO. 2, andthe tyrosine kinase domain has the amino acid sequence of SEQ ID NO. 3.In the amino acid sequence of SEQ ID NO. 4 including the amino acidsequences of the SH3 domain, SH2 domain and tyrosine kinase domain, theSH3 domain corresponds to the 7th to 70th amino acids, the SH2 domaincorresponds to the 81st to 155th amino acids, and the tyrosine kinasedomain corresponds to the 192nd to 437th amino acids. Further, in theamino acid sequence of SEQ ID NO. 5 also including the amino acidsequences of the SH3 domain, SH2 domain and tyrosine kinase domain, theSH3 domain corresponds to the 48th to 111th amino acids, the SH2 domaincorresponds to the 122nd to 196th amino acids, and the tyrosine kinasedomain corresponds to the 233rd to 478th amino acids. SEQ ID NO. 6 is anexample of the nucleotide sequences of cDNAs coding for the polypeptidehaving the amino acid sequence of SEQ ID NO. 1. SEQ ID NO. 7 is anexample of the nucleotide sequences of cDNAs coding for the polypeptidehaving the amino acid sequence of SEQ ID NO. 2. SEQ ID NO. 8 is anexample of the nucleotide sequences of cDNAs coding for the polypeptidehaving the amino acid sequence of SEQ ID NO. 3.

It is known that the function of an SH2 domain is to recognize a proteincontaining a phosphorylated tyrosine residue and bind to thephosphorylated tyrosine residue (see Songyang et al., Cell 72: 767,1993), and the function of an SH3 domain is to recognize a proline-richregion of exchange factor SOS for a proto-oncogene, such as ras, andbind to the proline-rich region (see Egan et al., Nature 363: 45, 1993).It is considered that these individual SH2 and SH3 domains alone, or apeptide artificially prepared by combining these SH2 and SH3 domains,has a similar function to the function of the SH2 domain or/and the SH3domain. For evaluating the activity of the tyrosine kinase, it is usefuland important to use the following DNA fragments and RNA fragments whichare prepared from the respective nucleotide sequences coding for the SH2domain and the SH3 domain: a sense DNA, an anti-sense DNA, a sense RNA,an anti-sense RNA, or a derivative thereof, such as one which isobtained by methylation, methylphosphorylation, deamination orthiophosphorylation of the above sense DNA, anti-sense DNA, sense RNA oranti-sense RNA.

The amino acid sequence of SEQ ID NO. 5 is highly homologous to that ofCSK which is a known cytoplasmic tyrosine kinase having an SH2 domain,an SH3 domain and a kinase domain (with respect to CSK, see Nada et al.,Nature 351: 60, 1991). The homology between the amino acid sequence ofSEQ ID NO. 5 and the amino acid sequence of CSK is 53.9% with respect tothe entire amino acid sequence of tyrosine kinase, 57.4% with respect tothe SH2 domain only, 40.5% with respect to the SH3 domain only, and58.9% with respect to the kinase domain only. CSK has the activity tospecifically phosphorylate the C-terminal tyrosine residues ofexpression products of src family genes to thereby suppress activationof the expression products of the src family genes. Recent study of micedeficient in CSK gene, which are generated by gene targeting, showsthat, when the expression products of src family genes are constantlyactivated in mice, all mice die at their early embryonic stages, due todefects in the formation of neural tubes (see Imamoto & Soriano, Cell73: 1117, 1993; and Noda et al., Cell 73: 1125, 1993). Expression of theCSK gene is found in various organs. However, in view of the fact thatthe expression of the gene coding for the tyrosine kinase of the presentinvention is found only in brain cells and only at the earlydifferentiation stage of blood cells (see Example 8 below), the tyrosinekinase of the present invention appears to play a physiologicallydifferent role from the role of CSK.

Tyrosine kinases can generally phosphorylate the tyrosine residues of anenolase protein and a synthetic peptide as substrates, both of whichhave a tyrosine-rich sequence. However, with respect to substrates uponwhich tyrosine kinases act in cells, there are different substratesspecific for respective tyrosine kinases. Therefore, it is consideredthat a substrate for the novel tyrosine kinase of the present inventionis also specific and different from the substrates for theconventionally known tyrosine kinases. Further, recent study on an SH3domain and an SH2 domain using a synthetic peptide library containingphosphorylated tyrosines shows that the SH3 domain and the SH2 domainindividually, specifically recognize certain amino acid sequences andbind to them. Therefore, it is considered that not only the entireregion of the tyrosine kinase of the present invention, but also the SH3domain, the SH2 domain and the tyrosine kinase domain thereof haverespective specific substrates and binding proteins therefor, which aredifferent from those for the conventionally known tyrosine kinases.

It is known that an mRNA having a plurality of initiation codons (AUG)may be translated into various polypeptide fragments despite a fixednucleotide sequence thereof, as mentioned in general remarks of theliterature by M. Kozak (J. Cell Biol. 115: 887, 1991). In the presentinvention, the cDNA of SEQ ID NO. 11 has two initiation codonsrespectively at a position of the 208th to 210th nucleotides and at aposition of the 331st to 333rd nucleotides, so that two differentpolypeptide fragments shown in SEQ ID NOs. 5 and 4 can be obtained byexpression of the cDNA. SEQ ID NO. 9 is an example of cDNA sequencescoding for a polypeptide having the amino acid sequence of SEQ ID NO. 4.SEQ ID NO. 10 is an example of cDNA sequences coding for a polypeptidehaving the amino acid sequence of SEQ ID NO. 5.

In the present invention, respective homologous variants of thepolypeptides having the amino acid sequences of SEQ ID NOs. 4 and 5 canbe prepared by standard methods without impairing tyrosine kinaseactivity.

In the present invention, the respective DNAs coding for thepolypeptides having the amino acid sequences of SEQ ID NOs. 4 and 5, andthe DNA of SEQ ID NO. 11, can take various forms, such as a cDNA, achromosomal DNA having introns and exons, and an artificial DNA which isobtained by ligating, to the cDNA or chromsomal DNA, synthesizedoligonucleotides prepared by a known DNA synthesis method. It is knownthat, by the degeneracy of the genetic code, a certain polypeptide canbe coded for by different nucleotide sequences. It is also known thatthe 5' non-coding region and the 3' non-coding region of a DNA do notparticipate in formation of the amino acid sequence of a polypeptidecoded for by the DNA, so that these non-coding regions are likely toundergo mutation.

As is apparent from Example 8 described below, the amount of mRNA of thegene coding for the tyrosine kinase of the present invention is small inundifferentiated UT-7, but is increased in accordance with thedifferentiation of UT-7 into megakaryocyte or erythroblast. Therefore,it is considered that the tyrosine kinase of the present invention playsan important role in the differentiation of blood cells.

As a method of preparing a replicable recombinant DNA moleculecomprising a replicable expression vector and, operably inserted in thevector, a part or whole of the DNA coding for the tyrosine kinase of thepresent invention, and as a method of transforming a microorganism oranimal cells with the above replicable recombinant DNA molecule,reference can be made, for example, to the methods in Example 10described below. The microorganisms or animal cells transformed with agene coding for the tyrosine kinase of the present invention can beutilized in fundamental studies of tyrosine kinases, studies of drugdesign, and development of pharmaceuticals. Examples of suchpharmaceuticals include a carcinostatic agent which specificallycontrols a signal transmission system mediated by the tyrosine kinase,and an agent for controlling the differentiation of blood cells.

The anti-sense DNA and the anti-sense RNA of the present invention,which are prepared utilizing a part of the nucleotide sequence of SEQ IDNO. 11, can be used in studies of the differentiation of blood cells,and development of pharmaceuticals, such as an agent for controlling thedifferentiation of blood cells. It is also possible to regulate geneexpression in cells by use of a DNA fragment or an RNA fragment, whichis complementary to or corresponding to at least a part of thenucleotide sequence of SEQ ID NO. 11, such as a sense DNA, an anti-senseDNA, a sense RNA, an anti-sense RNA, or a derivative thereof which isobtained by methylation, methylphosphorylation, deamination orthiophosphorylation of the sense DNA, anti-sense DNA, sense RNA oranti-sense RNA. As is shown in Example 8 below, the anti-sense DNA hasthe ability to specifically bind to the mRNA of the gene coding for thetyrosine kinase of the present invention and, therefore, the anti-senseDNA can be advantageously used for detecting the expression of thetyrosine kinase gene of the present invention. In addition, as is shownin Example 9 below, the anti-sense RNA can also be advantageously usedin a likewise manner for detecting the expression of the tyrosine kinasegene of the present invention. The size of each of the sense DNA, theanti-sense DNA, the sense RNA, the anti-sense RNA and the derivativesthereof is at least 12 mer, preferably 14 mer to 16 mer.

Example 11 (which is described below) shows an example of methods ofpreparing an antibody using a polypeptide comprising as an immunogen atleast part of an amino acid sequence of the tyrosine kinase of thepresent invention. The prepared antibody can be advantageously used forvarious purposes, such as detection of the tyrosine kinase of thepresent invention.

According to the present invention, a chemical substance having thecapability to inhibit or activate the tyrosine kinase activity definedabove can be obtained by screening, utilizing the above-mentionedcapability as a criterion. More specifically, it is possible to screen achemical substance having the capability to inhibit or activate thetyrosine kinase activity of at least the tyrosine kinase of the presentinvention, utilizing the above-mentioned capability as a criterion. Itis also possible to obtain a chemical substance having the capability toregulate the intracellular signal transmission mediated by the tyrosinekinase of the present invention by screening, wherein inhibition of thefunctions of the SH2 domain and the SH3 domain of the tyrosine kinase ofthe present invention to recognize and bind to their respectiverecognition sites is utilized as a criterion for the detection (asmentioned above, SH2 specifically recognizes a phosphorylated tyrosineresidue of a protein, and SH3 specifically recognizes the proline-richregion of exchange factor SOS for a proto-oncogene, such as ras). Forexample, as shown in Example 13 below, erbstatin having the capabilityto inhibit tyrosine kinase activity (with respect to erbstatin, seeUmezawa et al., J. Antibiot. 39: 170, 1986) can be detected and isolatedby screening, wherein inhibition of tyrosine kinase activity is utilizedas a criterion for the detection. It is expected that a novel substancewould be able to be isolated by this method. This method can also beutilized for development of useful pharmaceuticals, such as acarcinostatic agent, and an agent for inhibiting or promoting theproliferation of a cell.

The cDNA coding for the tyrosine kinase of the present invention can beobtained from cell lines other than UT-7, such as human peripheral bloodmononuclear cells (such as T cells and NK cells), human acutemyelogenous leukemia cell line KGla (see Blood 62: 709, 1983), cell lineKMT-2 established from human umbilical cord blood (see Blood 76: 501,1990), and human chronic myelogenous leukemia cell line K562 (see Blood45: 321, 1975) (ATCC accession No. CCL243), in substantially the samemanner as in Examples 1 to 7.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be illustrated with reference toExamples, which however should not be construed as limiting the scope ofthe present invention.

EXAMPLE 1

Preparation of a poly(A)⁺ RNA of human blood cell line UT-7!

Cell line UT-7 (Komatsu et al., Cancer Res. 51: 341, 1991) (UT-7 isavailable from Dr. Norio Komatsu, instructor at the Department ofHematology, Jichi Medical School, Japan) is cultured and passaged by amethod in which human granulocyte monocyte colony-stimulating factor(hGM-CSF) is added to Iscove's modified Dulbecco's medium (IMDM)containing 10% fetal calf serum (FCS) so that the concentration ofhGM-CSF becomes 2 ng/ml, and UT-7 cells are inoculated in the resultantculture medium and incubated at 37° C. in a CO₂ incubator. Cultured UT-7cells are obtained.

The differentiation of UT-7 cells into megakaryocites are conducted inthe following manner. The cultured UT-7 cells in the culture medium arediluted with the same culture medium so that the concentration of thecells becomes 2×10⁵ cells/ml. Then, hGM-CSF is added to the resultantdiluted cells so that the concentration of hGM-CSF becomes 2 ng/ml, andPhorbol 12-Myristate 13-Acetate (PMA) is added thereto so that theconcentration of PMA becomes 10 ng/ml. Then, the cells are incubated for3 days, and the resultant megakaryocytes are recovered.

On the other hand, the differentiation of UT-7 cells into erythroblastsare conducted in the following manner. The cultured UT-7 cells in theculture medium are diluted with the same culture medium so that theconcentration of the cells becomes 2×10⁵ cells/ml. Human erythropoietin(hEPO) is added to the resultant diluted cells so that the concentrationof hEPO becomes 1 Unit/ml, and butyric acid is added thereto so that theconcentration of n-butyric acid becomes 1.3 mM. Then, the cells areincubated for 3 days, and the resultant erythroblasts are recovered.

With respect to each of these three types of cells, i.e., cells ofunstimulated UT-7, megakaryocytes and erythroblasts, 1×10⁸ cells arecollected and washed three times with PBS (-), and are used for theextraction of RNA in the following manner.

From the collected cells, total RNA is extracted by LithiumChloride/Urea method (Eur. J. Biochem. 107: 303, 1980). Then, a poly(A)⁺ RNA is isolated and purified from the total RNA by usingOligotex-dT 30 (manufactured and sold by Takara Shuzo Co., Ltd., Japan).

EXAMPLE 2

Construction of a primer specific for tyrosine kinase gene!

PCR is conducted according to the method of Wilks (Proc. Natl. Acad.Sci. USA 86: 1603, 1989), wherein use is made of two types of mixedprimers respectively having sequences complementary to subdomains 7 and9 of tyrosine kinase, and having their respective restriction sitesligated therein, that is, sense primer PTKI:5'-TTGTCGACAC(AC)G(AG)GA(CT)(CT)T(CG)GC(ACGT)GC(ACGT)(AC)G-3' (27-merhaving SalI site as a restriction site), and anti-sense primerPTKII:3'-CT(AG)CA(CG)ACC(AT)(CG)(AG)A(AT)ACCTTAAGGT-5' (24-mer havingEcoRI site as a restriction site), as follows.

A synthetic oligonucleotide is constructed by means of an automatic DNAsynthesizer using a solid phase method. As the automatic DNAsynthesizer, 391PCR-MATE (manufactured and sold by APPLIED BIOSYSTEMS,INC., U.S.A.) is used. Nucleotides, a carrier having 3'-nucleotidesupported thereby, a solution, and a reagent are used in accordance withthe manual accompanying the synthesizer. A coupling reaction isconducted to synthesize an oligonucleotide in a state supported by acarrier. A protecting group at the 5' terminus of the oligonucleotide isremoved by applying trichloroacetic acid to the oligonucleotide, and theoligonucleotide is placed in a concentrated ammonia solution and allowedto stand at room temperature for 1 hour, thereby liberating theoligonucleotide from the carrier. Subsequently, the concentrated ammoniasolution containing the oligonucleotide liberated from the carrier isallowed to stand at 55° C. for more than 14 hours in a sealed vial, tothereby remove the protecting groups from both the nucleic acid andphosphate of the oligonucleotide. The obtained oligonucleotide, fromwhich the carrier and protecting groups have been removed, is purifiedby means of an OPC cartridge (manufactured and sold by APPLIEDBIOSYSTEMS, INC., U.S.A.), and detritylated using 2% trifluoroaceticacid, thereby obtaining a purified primer. The purified primer isdissolved in deionized water so that the concentration of the primerbecomes 1 μg/μl, and the obtained solution is used in the PCR describedbelow.

EXAMPLE 3

Synthesis of a cDNA!

A cDNA is synthesized utilizing the poly(A)⁺ RNA obtained in Example 1.Specifically, 2 μg of the poly(A)⁺ RNA is dissolved in 12.3 μl ofdeionized water, thereby obtaining a solution. To the obtained solutionare added 2 μl of 10× buffer containing 500 mM KCl, 100 mM Tris-HCl (pH8.3), 15 mM MgCl₂, and 0.01% gelatin!, 4 μl of dNTP (2.5 mM), 1 μl ofthe above-mentioned anti-sense primer PTKII (1 μg/μl) specific fortyrosine kinase, 0.2 μl of avian myeloblastosis virus reversetranscriptase (manufactured and sold by Life Science Laboratories,U.S.A: 32 U/μl), and 0.5 μl of RNase inhibitor (manufactured and sold byBoehringer-Mannheim GmbH, Germany: 40 U/μl). The resultant mixture isallowed to stand at 37° C. for 75 minutes, and further allowed to standat 65° C. for 10 minutes.

EXAMPLE 4

PCR using primers specific for the tyrosine kinase gene!

Amplification of the cDNA by PCR is conducted as follows. To 20 μl ofthe cDNA solution obtained in Example 3 are added 8 μl of 10× buffercontaining 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and 0.01%gelatin!, 6.4 μl of dNTP (2.5 mM), 1.5 μl of the above-mentioned senseprimer PTKI (1 μg/μl) specific for tyrosine kinase, and 0.2 μl of TaqDNA polymerase (AmpliTaq: manufactured and sold by Perkin Elmer CetusCo., Ltd., U.S.A.: 5 U/μl). To the resultant mixture, deionized water isadded so that the total volume becomes 100 μl. PCR procedure isconducted first at 94° C. for 1 minute, next at 37° C. for 2 minutes,and then at 72° C. for 3 minutes. This procedure is repeated 40 timesand then, the reaction mixture is allowed to stand at 72° C. for 7minutes. A portion of the resultant PCR product is electrophoresed in 2%agarose gel. Subsequently, the gel is then stained with ethidium bromideand observed under ultraviolet light, to thereby confirm that a cDNAhaving about 210 bp nucleotides has been amplified.

EXAMPLE 5

Cloning and sequencing of the PCR product!

A whole amount of the remaining PCR product is electrophoresed in 2%agarose gel composed of low melting point agarose. Subsequently, the gelis stained with ethidium bromide, and a band corresponding to about 210bp is confirmed under ultraviolet light, and a gel portion having theband containing cDNA is cut out. The thus obtained cDNA is purified fromthe gel. The purified cDNA is digested with restriction enzymes EcoRIand SalI, and a portion of the digested cDNA is electrophoresed inagarose gel to obtain a cDNA fragment which is to be inserted into avector.

As a vector, pBluescript II KS (manufactured and sold by Toyobo Co.,Ltd., Japan) is employed. Before introducing the above prepared cDNAfragment into the vector, the vector is digested with restrictionenzymes EcoRI and SalI, and termini of the digested vector aredephosphorylated with alkaline phosphatase (CIAP: manufactured and soldby Takara Shuzo Co., Ltd., Japan). The obtained digested vector and theabove-mentioned cDNA are mixed with each other in a molar ratio of 1:5,and the cDNA is ligated into the vector by using T4 ligase (manufacturedand sold by New England Bio Labs, U.S.A.). The resultant vectorpBluescript having the cDNA inserted therein is introduced into E. colistrain JM109, and the strain is seeded on a plate of semi-solid L-brothmedium containing 50 μg/ml of ampicillin, and allowed to stand at 37° C.for 12 hours. From colonies which have appeared, colonies are selectedat random, and digested with restriction enzymes EcoRI and SalI tothereby confirm that a cDNA having about 210 bp can be cut out by thedigestion. Thus, it is confirmed that the desired cDNA has beenintroduced in the selected clones. The nucleotide sequence of the cDNAof each of the confirmed clones is determined by means of fluorescencesequencer model 373A (manufactured by APPLIED BIOSYSTEMS, INC., U.S.A.).As a result, it is found that, with respect to the clones having thecDNA prepared using the poly(A)+RNA derived from the unstimulated UT-7cells, the tyrosine kinase gene of the present invention is detectedfrom only 1 clone among 98 clones; with respect to the clones having thecDNA prepared using the poly(A)⁺ RNA derived from the megakaryocitesdifferentiated from the UT-7 cells, the tyrosine kinase gene of thepresent invention is detected from 8 clones among 51 clones; and withrespect to the clones having the cDNA prepared using the poly(A)⁺ RNAderived from the erythroblasts differentiated from the UT-7 cells, thetyrosine kinase gene of the present invention is detected from 7 clonesamong 53 clones. This indicates that the tyrosine kinase gene of thepresent invention does far more frequently occur in differentiated cellsthan in undifferentiated cells. This gene of the present inventioncorresponds to the 1276th to 1421st nucleotides of the nucleotidesequence of SEQ ID NO. 11.

EXAMPLE 6

Preparation of a cDNA library, and cloning of the entire region of thetyrosine kinase gene of the present invention!

Using the poly(A)⁺ RNA of the unstimulated UT-7 cells which has beenisolated and purified according to the above-described method, a cDNAlibrary is prepared. For the preparation of a cDNA library, pCDM8 vectorcDNA library construction kit (manufactured and sold by Invitrogen,Netherlands) is used in accordance with the manual accompanying the kit.

Next, by using colony hybridization method, a cDNA clone coding for theentire region of the tyrosine kinase gene of the present invention issearched from colonies containing about 5×10⁵ cells in the cDNA libraryprepared above. The hybridization is conducted in the following manner.The colonies of cells are transferred to a nylon filter (Hybond N+:manufactured and sold by Amersham International, U.K.). The nylon filterhaving the cells thereon is treated with alkali (by leaving the nylonfilter for 7 minutes on a filter paper which contains, absorbed therein,1.5M NaCl and 0.5M NaOH), neutralized (by leaving the nylon filter for 3minutes on a filter paper which contains, absorbed therein, 1.5M NaCl,0.5M Tris-HCl pH 7.2, and 1 mM EDTA) twice, washed by shaking for 5minutes in SSPE of 2-fold concentration (0.36M NaCl, 0.02M sodiumphosphate pH 7.7, 2 mM EDTA), and air dried. Then, the thus treatednylon filter is left for 20 minutes on a filter paper which contains0.4M NaOH absorbed therein, washed by shaking for 5 minutes in SSPE of5-fold concentration, and air dried. The dried filter is subjected toscreening by using a cDNA probe labeled with radioisotope ³² P Thescreening is conducted in the following manner.

A cDNA probe labeled with radioisotope ³² P is prepared as follows. ThepBluescript having, inserted therein, the cDNA fragment coding for thetyrosine kinase of the present invention, which is obtained in Example5, is digested with restriction enzymes SalI and EcoRI, to thereby cutout the cDNA fragment, and electrophoresed in low melting point agarose.Then, the cDNA fragment is separated and purified from the gel, and thepurified cDNA fragment is labeled by means of a DNA labeling kit(Megaprime DNA labeling system: manufactured and sold by AmershamInternational, U.K.). Specifically, the labeling is conducted asfollows. To 25 ng of the DNA fragment are added 5 μl of a primersolution and deionized water so that the total volume becomes 33 μl, andthe resultant mixture is heated in a boiling water bath for 5 minutes.To the mixture are then added 10 μl of a reaction buffer containingdNTP, 5 μl of α-³² P-dCTP, and 2 μl of a DNA polymerase solution, andthe resultant mixture is heated in a water bath at 37° C. for 10minutes. From the heated mixture, the DNA is purified using Sephadexcolumn (Quick Spin Column Sephadex G-50: manufactured and sold byBoehringer-Mannheim GmbH, Germany). The purified DNA is heated in aboiling water bath for 5 minutes, and cooled in an ice bath for 2minutes.

The above-mentioned nylon filter carrying cells to be screened isimmersed in a prehybridization solution composed of SSPE of a 5-foldconcentration, a Denhardt's solution of a 5-fold concentration, 0.5% SDS(sodium dodecyl sulfate), and 10 mg/ml of salmon sperm DNA denatured ina boiling water bath, wherein the concentration of each component is afinal concentration as determined in the prehybridization solution. Thefilter in the prehybridization solution is shaken at 65° C. for 2 hours.Hybridization is conducted by shaking the filter at 65° C. for 16 hoursin a hybridization solution which has the same composition as that ofthe above-mentioned prehybridization solution except that itadditionally contains the above-obtained ³² P-labeled cDNA probe.

The filter is then washed twice by immersing it with shaking in an SSPEsolution containing 0.1% SDS at 65° C., and further washed 4 times at65° C. in a solution obtained by 10-fold diluting an SSPE solutioncontaining 0.1% SDS with water. The washed filter is subjected toautoradiography by use of a sensitized screen. Clones are picked up froma strongly luminous colony, and seeded again to thereby grow colonies,and a screening is conducted in the same manner as described above,thereby obtaining a desired clone.

According to the method described in the above-mentioned laboratorymanual by Maniatis et al., plasmid pCDM8 is purified from the obtainedclones. The purified plasmid is digested with restriction enzyme XhoI tothereby obtain a cDNA. The cDNA is purified by electrophoresis in lowmelting point agarose, and the purified cDNA is inserted into plasmidpBluescript. The nucleotide length of the purified cDNA is about 2 kbp.

EXAMPLE 7

Determination of the nucleotide sequence of the tyrosine kinase gene ofthe present invention!

The cDNA coding for the tyrosine kinase of the present inventionobtained in Example 6 is sequenced using ALFDNA sequencer (manufacturedand sold by Pharmacia Fine Chemicals AB, Sweden) and labeling kit forALF sequencer (which labeling kit is manufactured and sold by PharmaciaFine Chemicals AB, Sweden) according to the manuals accompanying thesequencer and the kit. Specifically, a deletion mutant is prepared,using a deletion mutant kit for kilosequence (manufactured and sold byTakara Shuzo Co., Ltd., Japan), according to the manual accompanying thekit. Using the deletion mutant, the entire nucleotide sequence of thecDNA coding for the tyrosine kinase of the present invention isdetermined in both directions.

EXAMPLE 8

Detection of production of the mRNA, which codes for the tyrosine kinaseof the present invention, in various types of tissue cells by northernblotting!

For detection of the mRNA coding for the tyrosine kinase of the presentinvention in human tissue cells and blood cells, the following filtersare used: commercially available filters carrying mRNAs of various typesof tissue cells, specifically, Human Multiple Tissue Northern Blot,Human Multiple Tissue Northern Blot II, Human Fetal Multiple TissueNorthern Blot, and Human Brain Multiple Tissue Northern Blot (allmanufactured and sold by Clontech, U.S.A.), and a filter prepared by amethod in which the mRNA obtained by the method shown in Example 1 iselectrophoresed in agarose gel and transferred to Zeta-Prob(manufactured and sold by Bio-Rad laboratories, U.S.A.). First, the cDNAcoding for the entire region of the tyrosine kinase of the presentinvention is digested with SmaI, thereby obtaining a fragment of about800 bp. The obtained fragment is labeled with ³² P by use of theabove-mentioned DNA labeling kit (MegaPrime DNA labeling system,manufactured and sold by Amersham International, U.K.), and the labeledfragment is reacted with the mRNAs carried by the above-mentionedfilters, to thereby examine the presence or absence of mRNA coding forthe tyrosine kinase of the present invention.

As a result, it has been found that, with respect to tissue cells of anadult human, the mRNA coding for the tyrosine kinase of the presentinvention is not detected in the total mRNA of the cells of heart,placenta, liver, skeletal muscle, kidney, pancreas, prostate, testis,ovary and small intestine. Whereas, it has been found that the mRNAcoding for the tyrosine kinase of the present invention is detected inthe total mRNA of the cells of brain, spleen, colon, and in the totalmRNA of peripheral blood lymphocytes. It has also been found that, inthe total mRNA of lung and thymus cells, the mRNA coding for thetyrosine kinase of the present invention is detected, although theamount of the mRNA is small. On the other hand, it has been found that,with respect to tissue cells of a human fetus, the mRNA coding for thetyrosine kinase of the present invention is not detected in the totalmRNA of the cells of heart, lung, liver and kidney, but detected in thetotal mRNA of the cells of brain. Regarding adult human brain tissuecells, the mRNA coding for the tyrosine kinase of the present inventionis detected in the total mRNA of the cells of amygdala, caudate nucleus,corpus callosum, hippocampus, hypothalamus, substantia nigra,subthalamic nucleus, and thalamus.

The mRNA coding for the tyrosine kinase of the present invention isdetected also in the total mRNAs respectively of human acute myelogenousleukemia cell line KG1a (see Blood 62: 709, 1983), cell line KMT-2established from human umbilical cord blood (see Blood 76: 501, 1990),human chronic myelogenous leukemia cell line K562 (see Blood 45: 321,1975) (ATCC accession No. CCL243), and the above-mentioned myelogenousmegakaryoblastic leukemia cell line UT-7. Further, it has been foundthat, with respect to cell lines K562 and UT-7, the amount of the mRNAcoding for the tyrosine kinase of the present invention is increasedwhen they are induced to differentiate into megakaryocytes bystimulation with PMA.

Therefore, it is considered that the mRNA coding for the tyrosine kinaseof the present invention is specifically produced in brain cells andblood cells, and that the production of this mRNA in blood cells isincreased in accordance with the differentiation of the blood cells.

The mRNA coding for the tyrosine kinase of the present invention is notdetected in lymphocytic cells, such as hepatocellular carcinoma cellline Hep3B (Nature 282: 615, 1979), fetal pulmonary fibroblast lineMRC-5 (Nature 227: 168, 1970), and acute lymphocytic leukemia cell lineMOLT-4 (J. Nat. Cancer Inst. 49: 891, 1972).

EXAMPLE 9

Detection of production of the mRNA, which codes for the tyrosine kinaseof the present invention, in peripheral blood cells by in situhybridization technique!

The mRNA coding for the tyrosine kinase of the present invention inperipheral blood lymphocytes is detected as follows. Mononuclear cellsare isolated from adult human peripheral blood by use of Ficoll-Paque(trade mark) (manufactured and sold by Pharmacia Fine Chemicals AB,Sweden). The isolated mononuclear cells are fixed onto a slide glass bymeans of Cytospin 3 (manufactured and sold by Shandon inc., U.K.). Themononuclear cells are subjected to fixation-treatment for 15 minuteswith 4% paraformaldehyde PBS. Subsequently, the slide glass having thecells fixed thereto is immersed for 15 minutes in a solution containing10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 10 μg/ml pronase K. The cellsfixed to the slide glass are further subject to fixation-treatment for15 minutes with 4% paraformaldehyde PBS, washed once with PBS, immersedin 0.2M HCl for 10 minutes, washed once with PBS, and immersed in 0.1Mtriethanolamine-HCl (pH 8.0) for 1 minute. Subsequently, the cells areimmersed for 10 minutes in a solution containing 0.1Mtriethanolamine-HCl (pH 8.0) and 0.25% acetaldehyde, and washed withPBS. Then, the cells are successively immersed in 70% ethanol for 15seconds, 80% ethanol for 15 seconds, 90% ethanol for 15 seconds, and100% ethanol for 15 seconds, to thereby dehydrate the cells completely.Then, the dehydrated cells are air dried. All of the above-mentionedtreating solutions are those which have been cooled to 4° C. before use.The cells fixed by the above operation are used for the in situhybridization, which is described below.

A digoxigenin-labeled RNA probe for use in the in situ hybridization isprepared in the following manner. A SmaI fragment of the cDNA coding forthe entire region of the tyrosine kinase of the present invention, whichis the same fragment as used in the northern blotting in Example 8, isligated into the SmaI site of pBluescript having dephosphorylatedtermini, to thereby obtain a vector for in situ hybridization. Theobtained vector is purified using a plasmid purification column(manufactured and sold by QUIAGEN, Germany), and digested withrestriction enzymes BamHI and EcoRI. From nucleotide fragments obtainedby the digestion, an anti-sense RNA probe and a sense RNA probe (as acontrol) for detecting the mRNA coding for the tyrosine kinase of thepresent invention are prepared using T3 or T7 RNA polymerase, and DIGRNA labeling kit (manufactured and sold by Boehringer-Mannheim GmbH,Germany) according to the manual accompanying the kit. These probes areused for the detection of the mRNA coding for the tyrosine kinase of thepresent invention in peripheral blood mononuclear cells.

A hybridization solution containing 50% formamide, 10 mM Tris-HCl (pH7.6), 200 μg/ml of tRNA, 1-fold Denhardt's solution, 10% dextransulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA (pH 8.0)! is heated toand kept at 85° C. for 10 minutes. The sense RNA probes and theanti-sense RNA probes are individually added to the heated solution sothat the concentration of each of the RNA probes becomes 10 μg/ml, andeach of the resultant mixtures is further kept at 85° C. for 3 minutes,to thereby denature the RNA probes. The resultant hybridizationsolutions containing the denatured RNA probes are individually, dropwiseadded to the above-mentioned air-dried cells on the slide glass. Thecells are covered with Parafilm and allowed to stand at 50° C. for 16hours to thereby conduct a hybridization.

After completion of the hybridization, the cells on the slide glass areimmersed in 5× SSC at 50° C. to thereby remove the Parafilm from thecells, and then immersed in a mixture of 2× SSC and 50% formamide at 60°C. for 30 minutes. Then, the cells are immersed in a solution containing10 mM Tris-HCl (pH 7.6), 500 mM NaCl and 1 mM EDTA at 37° C. for 10minutes, immersed in a solution at 37° C. for 30 minutes, which solutionhas been prepared by adding RNase A to the same solution as mentionedabove so that the concentration of RNase A becomes 20 μg/ml, and furtherimmersed in the same solution as mentioned above, which does not containRNase A, at 37° C. for 10 minutes. Then, the cells are immersed in 2×SSC at 50° C. for 20 minutes, and further immersed in 0.2× SSC at 50° C.for 20 minutes twice, to thereby wash the cells.

Cells containing the mRNA coding for the tyrosine kinase of the presentinvention are stained using a DIG nucleic acid assay kit containing analkaline phosphatase-labeled DIG antibody (manufactured and sold byBoehringer-Mannheim GmbH, Germany) according to the manual accompanyingthe kit. Further, the cells are also stained with hematoxylin to therebymorphologically identify the cells containing the mRNA. As a result, itis confirmed that the mRNA coding for the tyrosine kinase of the presentinvention is produced in a large amount in lymphocytic cells.

In order to more specifically identify the lymphocytic cells containingthe mRNA coding for the tyrosine kinase of the present invention, humanperipheral blood mononuclear cells are stained, individually usingFITC-labeled anti-CD3 antibody (Leu 4, manufactured and sold by BECTONDICKINSON AND COMPANY, U.S.A.), FITC-labeled anti-CD19 antibody (Leu 12,manufactured and sold by BECTON DICKINSON AND COMPANY, U.S.A.), andFITC-labeled anti-CD56 antibody (Leu 19, manufactured and sold by BECTONDICKINSON AND COMPANY, U.S.A.), which antibodies are able tospecifically react with a T cell, a B cell and an NK cell, respectively.The stained mononuclear cells are classified into 3 groups by use offlow cytometer EPICS Elite (manufactured and sold by Coulter corp.,U.K.), according to the type of antibody with which the cells havereacted. With respect to the 3 groups of cells, the presence or absenceof the mRNA coding for the tyrosine kinase of the present invention isexamined by the method described above. As a result, it is found thatabout 5% to 20% of the cells which have reacted with anti-CD3 antibody,and about 30% to 70% of the cells which have reacted with anti-CD56antibody contain the mRNA coding for the tyrosine kinase of the presentinvention, whereas almost all of the cells which have reacted withanti-CD19 antibody do not contain the mRNA coding for the tyrosinekinase of the present invention. These results clearly show that thetyrosine kinase of the present invention plays an important role in thedifferentiation, proliferation and functioning of T cells and NK cells.

EXAMPLE 10

Preparation of a transformant cell strain having the tyrosine kinasegene of the present invention introduced therein!

DNAs respectively having the sequences of SEQ ID NOs. 9 and 10 areindividually synthesized by the PCR method, based on the nucleotidesequence of SEQ ID NO. 11, i.e., the tyrosine kinase gene of the presentinvention. An early promoter of SV 40 virus is ligated upstream of eachof the synthesized DNAs, and a termination codon (TGA in DNA sequence),a poly (A)⁺ signal of SV 40 virus and the dihydrofolic acid reductasegene are ligated, in this order, downstream of each of the synthesizedDNAs, to thereby prepare expression plasmid vectors for use inproduction of polypeptides comprising the amino acid sequences of SEQ IDNOs. 4 and 5, respectively.

Each of the prepared plasmids is purified. 20 μg of each of the purifiedplasmids is individually introduced to CHO cells suspended in a glucosesolution. Introduction of the plasmids into the CHO cells is conductedby applying 600 V electricity to the cell suspension, using a genepulser (manufactured and sold by Bio-Rad laboratories, U.S.A.). The thustreated CHO cells are cultured in a medium containing 10% fetal calfserum for 2 days. Then, the CHO cells are further cultured in a mediumDulbecco's MEM containing 10% dialyzed fetal calf serum and methotrexate(MTX)!, to thereby select cells which have been transformed. mRNA isextracted from the selected cells, and poly(A)⁺ RNA is isolated from themRNA by use of an oligo dT gel column. A filter for use in northernblotting is prepared by utilizing the poly(A)⁺ RNA according to themethod shown in Example 8. Using the filter, northern blotting isconducted in the same manner as in Example 8 to thereby confirm thepresence of the mRNA coding for the tyrosine kinase of the presentinvention. As a result, it is confirmed that the transformant cellscapable of producing the tyrosine kinase of the present invention isobtained.

DNAs respectively having the sequences of SEQ ID NOs. 6, 7 and 8 areindividually prepared by utilizing the PCR method. Each of the preparedDNAs is individually ligated to restriction site SmaI of an expressionvector pGEX-4T-2 (manufactured and sold by Pharmacia Fine Chemicals AB,Sweden) which can be used for producing a fused protein containingglutathione S transferase, to thereby obtain expression plasmid vectorswhich can be used for producing polypeptides comprising amino acidsequences of SEQ ID NOs. 1, 2 and 3, respectively. Each of the thusobtained expression plasmid vectors respectively containing thesequences of SEQ ID NOs. 6, 7 and 8 is individually introduced to E.Coli strain DH5α, and the resultant cells are cultured on a selectionmedium containing ampicillin, to thereby select transformant cellstrains having the above-mentioned expression plasmid vectors,respectively.

EXAMPLE 11

Production of the tyrosine kinase peptide of the present invention!

First, a polyclonal antibody to be used for confirmation of theproduction of the tyrosine kinase peptide of the present invention isproduced as follows. A peptide is synthesized, which has a structuresuch that cysteine is bonded to the N-terminus of a peptide comprised ofthe 488th to 507th amino acids of the amino acid sequence of SEQ ID NO.5 (which 488-507 amino acid sequence of SEQ ID NO. 5 corresponds to theC-terminal portion of the amino acid sequence deduced from thenucleotide sequence of the tyrosine kinase gene of the presentinvention). Specifically, a peptide, having the amino acid sequence: CysPro Ala Ser Val Ser Gly Gln Asp Ala Asp Gly Ser Thr Ser Pro Arg Ser GlnGlu Pro, is synthesized by means of a peptide synthesizer. The obtainedpeptide is coupled with KLH (Keyhole Limpet Hemocyanin) through a thiolgroup, thereby obtaining a KLH-coupled peptide. The KLH-coupled peptideis employed as an immunogen. Specifically, the KLH-coupled peptide ismixed with Freund's adjuvant, and a rabbit is immunized with theresultant mixture. From the immunized rabbit, antiserum is obtained.

Each of the transformant cell strains obtained in Example 10 which canrespectively produce polypeptides having the amino acid sequences of SEQID NOs. 4 and 5 is individually solubilized with RIPA buffer containing0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 20 mM Tris-HCl (PH7.4), 1 mM PMSF, and 0.2 U/ml aprotinin!, and the protein content of thecell is quantitatively determined. Then, the cell-derived protein issubjected to a thermal denaturation with SDS and then to anSDS-polyacrylamide gel electrophoresis (SDS-PAGE). After completion ofthe electrophoresis, the protein is blotted against a PVDF filter(manufactured and sold by Bio-Rad laboratories, USA), and the resultantprotein-blotted filter is reacted with each of the above-mentionedantiserum and serum obtained from the rabbit prior to the immunization,individually, and then reacted with a peroxidase-labeled mousemonoclonal antibody against rabbit antibody. Subsequently, the proteinblotted on the filter, which protein has been reacted with theperoxidase-labeled monoclonal antibody, is allowed to emit fluorescenceby means of ECL Western Blot Detection System (manufactured and sold byAmersham International, U.K.) and the fluorescence was recorded on aphotosensitive film, thereby detecting the protein. By comparing theresults obtained with respect to the antiserum with the results obtainedwith respect to the prior-to-immunization serum, it is confirmed thatthe tyrosine kinase polypeptide of the present invention, having amolecular weight of about 55 kilodalton, has been produced.

The cell strains obtained in Example 10 which can respectively producepolypeptides having amino acid sequences of SEQ ID NOs. 1, 2 and 3 areindividually inoculated in a liquid medium for E. Coli, which containsampicillin, and incubated overnight. Subsequently, the incubated cellsare diluted 10-fold with the same liquid medium and further incubatedfor 2 hours, and IPTG is added thereto so that the final concentrationof IPTG becomes 1 mM, followed by incubation for 5 hours, therebyinducing production of protein. Subsequently, the cells are collected bycentrifugation, and suspended in PBS containing 1% Triton X-100. Thesuspended cells are disrupted by means of an ultrasonic cell disruptingapparatus (manufactured and sold by Taitec Corporation, Japan) whilecooling with ice, and the disrupted cells are subjected tocentrifugation, thereby obtaining a supernatant. The supernatant issubjected to SDS-PAGE in the same manner as mentioned above, therebyobtaining a protein. The obtained protein is blotted on a PVDF filterand reacted with an anti-GST goat antibody (manufactured and sold byPharmacia Fine Chemicals AB, Sweden) and further reacted with aperoxidase-labeled rabbit antibody against goat antibody, and thedetection of the protein is conducted in the same manner as mentionedabove by using ECL Western Blot Detection System. As a result, it isconfirmed that the cell strains have respectively produced a peptidehaving a molecular weight of about 33 kilodalton and having an aminoacid sequence of SEQ ID NO. 1, a peptide having a molecular weight ofabout 35 kilodalton and having an amino acid sequence of SEQ ID NO. 2,and a peptide having a molecular weight of about 63 kilodalton andhaving an amino acid sequence of SEQ ID NO. 3. Next, these three typesof GST-fused tyrosine kinase peptides are separated and purified fromthe disrupted cells by using a glutathione Sepharose column(manufactured and sold by Pharmacia Fine Chemicals AB, Sweden) inaccordance with the manual accompanying the column. The thus obtainedpurified peptides are subjected to SDS-PAGE and Coomassie staining, tothereby confirm the respective molecular weights of these peptidesdetermined by the above western blotting.

EXAMPLE 12

Confirmation of the enzyme activity of the tyrosine kinase of thepresent invention!

Each of the transformant cell strains obtained in Example 10 which canrespectively produce polypeptides having the amino acid sequences of SEQID NOs. 4 and 5 is individually placed in a 1.5 ml-volume Eppendorftube, and RIPA buffer is added to the cells to thereby solubilize thecells. Protein G Sepharose gel (manufactured and sold by Pharmacia FineChemicals AB, Sweden) is added to the solubilized cells, and theresultant mixture is gently stirred at 4° C. overnight to thereby effecta reaction. Subsequently, the resultant reaction mixture is subjected tocentrifugation, thereby precipitating protein G Sepharose gel andsubstances which have reacted nonspecifically to the protein G, followedby removal thereof. Thus, a disrupted cell suspension containing thetyrosine kinase polypeptide of the present invention is obtained. Theanti-tyrosine kinase polyclonal antibody obtained in Example 11 is addedto the disrupted cell suspension, and a reaction is conducted at 4° C.for 1 hour. Subsequently, protein G Sepharose gel is added thereto andgently stirred at 4° C. for 1 hour to thereby effect a reaction. Then, acentrifugation is conducted to thereby cause the protein G Sepharose gelwhich has adsorbed the tyrosine kinase of the present invention to beseparated as a precipitated fraction. The separated fraction is washedwith the above-mentioned RIPA buffer, and then subjected tocentrifugation in the same manner as described above. This operation isconducted three times. Thus, the tyrosine kinase polypeptide of thepresent invention which has been adsorbed on protein G Sepharose gel isobtained.

With respect to each of the above-obtained tyrosine kinase polypeptideadsorbed on the gel and the polypeptide obtained in Example 11comprising the amino acid sequence of SEQ ID NO. 3, the enzyme activityof tyrosine kinase is evaluated using a tyrosine kinase assay kit whichis of a non-radioreactive type (manufactured and sold byBoehringer-Mannheim GmbH, Germany). The assay is conducted in accordancewith the manual accompanying the kit. As a result, it is confirmed thatboth of the polypeptides have enzyme activity of tyrosine kinase.

EXAMPLE 13

Screening of chemical substances by utilizing the tyrosine kinase of thepresent invention!

Using the tyrosine kinase polypeptide of the present invention which hasbeen purified by the method described in Example 12 and the tyrosinekinase polypeptide of the present invention which has been purified bythe method described in Example 11, a substance having the capability toinhibit the tyrosine kinase activity of the tyrosine kinase of thepresent invention is searched with respect to supernatants of culturesof various Actinomycete cell lines, utilizing the above-mentionedcapability as a criterion for detection, and the detected substance isisolated and purified from the culture supernatant, and identified.

Specifically, about 1,000 cell lines of Actinomycetes are individuallycultured to obtain culture supernatants. The supernatants areindividually subjected to filtration to obtain filtrates, and anequivolume of butyl acetate is added to each supernatant to therebyeffect extraction. The extracted 1,000 types of substances areindividually dried in vacuo to obtain the substances in a powdery form.

Substances which inhibit tyrosine kinase activity are screened from theabove-obtained powdery substances by an assay method in which thetyrosine kinase assay kit described in Example 12 and either of thepolypeptides respectively having the sequences of SEQ ID NOs. 4 and 5are used to find out substances which lower the absorbance measured withrespect to the tyrosine kinase polypeptide of the present invention.More illustratively, the obtained 1,000 different powdery substances aretested with respect to the capability to lower the absorbance 30% ormore, based on the absorbance as measured with respect to the tyrosinekinase polypeptide without adding the powdery substance. As a result,about 50 different powdery substances are found to have the capability.

Of those substances, one powdery substance is selected, and aliquots ofthe selected powdery substance are dissolved in chloroform, methanol anddistilled water, respectively. The resultant solutions are individuallysubjected to filtration, and the resultant filtrates are individuallydried in vacuo to thereby obtain powdery materials. Each of the obtainedpowdery materials is assayed in the same manner as mentioned above and,as a result, it is confirmed that the powdery material derived from themethanol-solubles has an inhibitory activity for the tyrosine kinase ofthe present invention. The methanol-solubles are fractionated in areverse phase column by high-speed liquid chromatography. A peak of eachof the these fractions is collected using a fraction collector. Each ofthe collected fractions is further assayed in the same manner asmentioned above and, as a result, it is found that one of the fractionshas an inhibitory activity for the tyrosine kinase of the presentinvention. The fraction is dried in vacuo to thereby obtain a powder,and the powder is analyzed by mass spectography, NMR and IR. As aresult, it is found that the molecular formula of the substance of theabove fraction is C₉ H₉ NO₃. When a data base of chemical substances issearched to identify the substance, based on the chemical shiftsobtained by NMR and the spectra obtained by IR, it is found that thesubstance is identical to erbstatin (see Umezawa et al., J. Antibiot.39: 170, 1986). Erbstatin is known as a specific inhibitor for atyrosine kinase. These results show that the method of screeningchemical substances using the tyrosine kinase of the present inventionis effective for detecting useful chemical substances having thecapability to inhibit or activate tyrosine kinase activity.

INDUSTRIAL APPLICABILITY

The tyrosine kinase of the present invention can be advantageously usedfor screening chemical substances having the capability to inhibit oractivate the tyrosine kinase activity of at least the tyrosine kinase ofthe present invention. Further, the tyrosine kinase of the presentinvention and the gene coding therefor can be advantageously used forestimating or controlling the differentiation of blood cells. The genecoding for the tyrosine kinase of the present invention can be utilizedfor evaluation of various pharmaceuticals and for culturing blood stemcells without causing differentiation thereof. Further, it is expectedthat the gene coding for the tyrosine kinase of the present inventioncan be utilized for development of a carcinostatic agent and for genetherapy.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino acids                                                    (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaProGlyThrGlnCysIleThrLysCysGluHisThrArgProLys                              151015                                                                        ProGlyGluLeuAlaPheArgLysGlyAspValValThrIleLeuGlu                              202530                                                                        AlaCysGluAsnLysSerTrpTyrArgValLysHisHisThrSerGly                              354045                                                                        GlnGluGlyLeuLeuAlaAlaGlyAlaLeuArgGluArgGluAlaLeu                              505560                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 amino acids                                                    (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TrpPheHisGlyLysIleSerGlyGlnGluAlaValGlnGlnLeuGln                              151015                                                                        ProProGluAspGlyLeuPheLeuValArgGluSerAlaArgHisPro                              202530                                                                        GlyAspTyrValLeuCysValSerPheGlyArgAspValIleHisTyr                              354045                                                                        ArgValLeuHisArgAspGlyHisLeuThrIleAspGluAlaValPhe                              505560                                                                        PheCysAsnLeuMetAspMetValGluHisTyr                                             657075                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 246 amino acids                                                   (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlnHisLeuThrLeuGlyAlaGlnIleGlyGluGlyGluPheGlyAla                              151015                                                                        ValLeuGlnGlyGluTyrLeuGlyGlnLysValAlaValLysAsnIle                              202530                                                                        LysCysAspValThrAlaGlnAlaPheLeuAspGluThrAlaValMet                              354045                                                                        ThrLysMetGlnHisGluAsnLeuValArgLeuLeuGlyValIleLeu                              505560                                                                        HisGlnGlyLeuTyrIleValMetGluHisValSerLysGlyAsnLeu                              65707580                                                                      ValAsnPheLeuArgThrArgGlyArgAlaLeuValAsnThrAlaGln                              859095                                                                        LeuLeuGlnPheSerLeuHisValAlaGluGlyMetGluTyrLeuGlu                              100105110                                                                     SerLysLysLeuValHisArgAspLeuAlaAlaArgAsnIleLeuVal                              115120125                                                                     SerGluAspLeuValAlaLysValSerAspPheGlyLeuAlaLysAla                              130135140                                                                     GluArgLysGlyLeuAspSerSerArgLeuProValLysTrpThrAla                              145150155160                                                                  ProGluAlaLeuLysHisGlyLysPheThrSerLysSerAspValTrp                              165170175                                                                     SerPheGlyValLeuLeuTrpGluValPheSerTyrGlyArgAlaPro                              180185190                                                                     TyrProLysMetSerLeuLysGluValSerGluAlaValGluLysGly                              195200205                                                                     TyrArgMetGluProProGluGlyCysProGlyProValHisValLeu                              210215220                                                                     MetSerSerCysTrpGluAlaGluProAlaArgArgProProPheArg                              225230235240                                                                  LysLeuAlaGluLysLeu                                                            245                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 466 amino acids                                                   (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetProThrArgArgTrpAlaProGlyThrGlnCysIleThrLysCys                              151015                                                                        GluHisThrArgProLysProGlyGluLeuAlaPheArgLysGlyAsp                              202530                                                                        ValValThrIleLeuGluAlaCysGluAsnLysSerTrpTyrArgVal                              354045                                                                        LysHisHisThrSerGlyGlnGluGlyLeuLeuAlaAlaGlyAlaLeu                              505560                                                                        ArgGluArgGluAlaLeuSerAlaAspProLysLeuSerLeuMetPro                              65707580                                                                      TrpPheHisGlyLysIleSerGlyGlnGluAlaValGlnGlnLeuGln                              859095                                                                        ProProGluAspGlyLeuPheLeuValArgGluSerAlaArgHisPro                              100105110                                                                     GlyAspTyrValLeuCysValSerPheGlyArgAspValIleHisTyr                              115120125                                                                     ArgValLeuHisArgAspGlyHisLeuThrIleAspGluAlaValPhe                              130135140                                                                     PheCysAsnLeuMetAspMetValGluHisTyrSerLysAspLysGly                              145150155160                                                                  AlaIleCysThrLysLeuValArgProLysArgLysHisGlyThrLys                              165170175                                                                     SerAlaGluGluGluLeuAlaArgAlaGlyTrpLeuLeuAsnLeuGln                              180185190                                                                     HisLeuThrLeuGlyAlaGlnIleGlyGluGlyGluPheGlyAlaVal                              195200205                                                                     LeuGlnGlyGluTyrLeuGlyGlnLysValAlaValLysAsnIleLys                              210215220                                                                     CysAspValThrAlaGlnAlaPheLeuAspGluThrAlaValMetThr                              225230235240                                                                  LysMetGlnHisGluAsnLeuValArgLeuLeuGlyValIleLeuHis                              245250255                                                                     GlnGlyLeuTyrIleValMetGluHisValSerLysGlyAsnLeuVal                              260265270                                                                     AsnPheLeuArgThrArgGlyArgAlaLeuValAsnThrAlaGlnLeu                              275280285                                                                     LeuGlnPheSerLeuHisValAlaGluGlyMetGluTyrLeuGluSer                              290295300                                                                     LysLysLeuValHisArgAspLeuAlaAlaArgAsnIleLeuValSer                              305310315320                                                                  GluAspLeuValAlaLysValSerAspPheGlyLeuAlaLysAlaGlu                              325330335                                                                     ArgLysGlyLeuAspSerSerArgLeuProValLysTrpThrAlaPro                              340345350                                                                     GluAlaLeuLysHisGlyLysPheThrSerLysSerAspValTrpSer                              355360365                                                                     PheGlyValLeuLeuTrpGluValPheSerTyrGlyArgAlaProTyr                              370375380                                                                     ProLysMetSerLeuLysGluValSerGluAlaValGluLysGlyTyr                              385390395400                                                                  ArgMetGluProProGluGlyCysProGlyProValHisValLeuMet                              405410415                                                                     SerSerCysTrpGluAlaGluProAlaArgArgProProPheArgLys                              420425430                                                                     LeuAlaGluLysLeuAlaArgGluLeuArgSerAlaGlyAlaProAla                              435440445                                                                     SerValSerGlyGlnAspAlaAspGlySerThrSerProArgSerGln                              450455460                                                                     GluPro                                                                        465                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 507 amino acids                                                   (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetAlaGlyArgGlySerLeuValSerTrpArgAlaPheHisGlyCys                              151015                                                                        AspSerAlaGluGluLeuProArgValSerProArgPheLeuArgAla                              202530                                                                        TrpHisProProProValSerAlaArgMetProThrArgArgTrpAla                              354045                                                                        ProGlyThrGlnCysIleThrLysCysGluHisThrArgProLysPro                              505560                                                                        GlyGluLeuAlaPheArgLysGlyAspValValThrIleLeuGluAla                              65707580                                                                      CysGluAsnLysSerTrpTyrArgValLysHisHisThrSerGlyGln                              859095                                                                        GluGlyLeuLeuAlaAlaGlyAlaLeuArgGluArgGluAlaLeuSer                              100105110                                                                     AlaAspProLysLeuSerLeuMetProTrpPheHisGlyLysIleSer                              115120125                                                                     GlyGlnGluAlaValGlnGlnLeuGlnProProGluAspGlyLeuPhe                              130135140                                                                     LeuValArgGluSerAlaArgHisProGlyAspTyrValLeuCysVal                              145150155160                                                                  SerPheGlyArgHisValIleHisTyrArgValLeuHisArgAspGly                              165170175                                                                     HisLeuThrIleAspGluAlaValPhePheCysAsnLeuMetAspMet                              180185190                                                                     ValGluHisTyrSerLysAspLysGlyAlaIleCysThrLysLeuVal                              195200205                                                                     ArgProLysArgLysHisGlyThrLysSerAlaGluGluGluLeuAla                              210215220                                                                     ArgAlaGlyTrpLeuLeuAsnLeuGlnHisLeuThrLeuGlyAlaGln                              225230235240                                                                  IleGlyGluGlyGluPheGlyAlaValLeuGlnGlyGluTyrLeuGly                              245250255                                                                     GlnLysValAlaValLysAsnIleLysCysAspValThrAlaGlnAla                              260265270                                                                     PheLeuAspGluThrAlaValMetThrLysMetGlnHisGluAsnLeu                              275280285                                                                     ValArgLeuLeuGlyValIleLeuHisGlnGlyLeuTyrIleValMet                              290295300                                                                     GluHisValSerLysGlyAsnLeuValAsnPheLeuArgThrArgGly                              305310315320                                                                  ArgAlaLeuValAsnThrAlaGlnLeuLeuGlnPheSerLeuHisVal                              325330335                                                                     AlaGluGlyMetGluTyrLeuGluSerLysLysLeuValHisArgAsp                              340345350                                                                     LeuAlaAlaArgAsnIleLeuValSerGluAspLeuValAlaLysVal                              355360365                                                                     SerAspPheGlyLeuAlaLysAlaGluArgLysGlyLeuAspSerSer                              370375380                                                                     ArgLeuProValLysTrpThrAlaProGluAlaLeuLysHisGlyLys                              385390395400                                                                  PheThrSerLysSerAspValTrpSerPheGlyValLeuLeuTrpGlu                              405410415                                                                     ValPheSerTyrGlyArgAlaProTyrProLysMetSerLeuLysGlu                              420425430                                                                     ValSerGluAlaValGluLysGlyTyrArgMetGluProProGluGly                              435440445                                                                     CysProGlyProValHisValLeuMetSerSerCysTrpGluAlaGlu                              450455460                                                                     ProAlaArgArgProProPheArgLysLeuAlaGluLysLeuAlaArg                              465470475480                                                                  GluLeuArgSerAlaGlyAlaProAlaSerValSerGlyGlnAspAla                              485490495                                                                     AspGlySerThrSerProArgSerGlnGluPro                                             500505                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 192 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCCCCGGGCACCCAGTGTATCACCAAATGCGAGCACACCCGCCCCAAGCCAGGGGAGCTG60                GCCTTCCGCAAGGGCGACGTGGTCACCATCCTGGAGGCCTGCGAGAACAAGAGCTGGTAC120               CGCGTCAAGCACCACACCAGTGGACAGGAGGGGCTGCTGGCAGCTGGGGCGCTGCGGGAG180               CGGGAGGCCCTC192                                                               (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 225 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TGGTTCCACGGGAAGATCTCGGGCCAGGAGGCTGTCCAGCAGCTGCAGCCTCCCGAGGAT60                GGGCTGTTCCTGGTGCGGGAGTCCGCGCGCCACCCCGGCGACTACGTCCTGTGCGTGAGC120               TTTGGCCGCGACGTCATCCACTACCGCGTGCTGCACCGCGACGGCCACCTCACAATCGAT180               GAGGCCGTGTTCTTCTGCAACCTCATGGACATGGTGGAGCATTAC225                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 738 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CAGCATTTGACATTGGGAGCACAGATCGGAGAGGGAGAGTTTGGAGCTGTCCTGCAGGGT60                GAGTACCTGGGGCAAAAGGTGGCCGTGAAGAATATCAAGTGTGATGTGACAGCCCAGGCC120               TTCCTGGACGAGACGGCCGTCATGACGAAGATGCAACACGAGAACCTGGTGCGTCTCCTG180               GGCGTGATCCTGCACCAGGGGCTGTACATTGTCATGGAGCACGTGAGCAAGGGCAACCTG240               GTGAACTTTCTGCGGACCCGGGGTCGAGCCCTCGTGAACACCGCTCAGCTCCTGCAGTTT300               TCTCTGCACGTGGCCGAGGGCATGGAGTACCTGGAGAGCAAGAAGCTTGTGCACCGCGAC360               CTGGCCGCCCGCAACATCCTGGTCTCAGAGGACCTGGTGGCCAAGGTCAGCGACTTTGGC420               CTGGCCAAAGCCGAGCGGAAGGGGCTAGACTCAAGCCGGCTGCCCGTCAAGTGGACGGCG480               CCCGAGGCTCTCAAACACGGGAAGTTCACCAGCAAGTCGGATGTCTGGAGTTTTGGGGTG540               CTGCTCTGGGAGGTCTTCTCATATGGACGGGCTCCGTACCCTAAAATGTCACTGAAAGAG600               GTGTCGGAGGCCGTGGAGAAGGGGTACCGCATGGAACCCCCCGAGGGCTGTCCAGGCCCC660               GTGCACGTCCTCATGAGCAGCTGCTGGGAGGCAGAGCCCGCCCGCCGGCCACCCTTCCGC720               AAACTGGCCGAGAAGCTG738                                                         (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1398 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATGCCAACGAGGCGCTGGGCCCCGGGCACCCAGTGTATCACCAAATGCGAGCACACCCGC60                CCCAAGCCAGGGGAGCTGGCCTTCCGCAAGGGCGACGTGGTCACCATCCTGGAGGCCTGC120               GAGAACAAGAGCTGGTACCGCGTCAAGCACCACACCAGTGGACAGGAGGGGCTGCTGGCA180               GCTGGGGCGCTGCGGGAGCGGGAGGCCCTCTCCGCAGACCCCAAGCTCAGCCTCATGCCG240               TGGTTCCACGGGAAGATCTCGGGCCAGGAGGCTGTCCAGCAGCTGCAGCCTCCCGAGGAT300               GGGCTGTTCCTGGTGCGGGAGTCCGCGCGCCACCCCGGCGACTACGTCCTGTGCGTGAGC360               TTTGGCCGCGACGTCATCCACTACCGCGTGCTGCACCGCGACGGCCACCTCACAATCGAT420               GAGGCCGTGTTCTTCTGCAACCTCATGGACATGGTGGAGCATTACAGCAAGGACAAGGGC480               GCTATCTGCACCAAGCTGGTGAGACCAAAGCGGAAACACGGGACCAAGTCGGCCGAGGAG540               GAGCTGGCCAGGGCGGGCTGGTTACTGAACCTGCAGCATTTGACATTGGGAGCACAGATC600               GGAGAGGGAGAGTTTGGAGCTGTCCTGCAGGGTGAGTACCTGGGGCAAAAGGTGGCCGTG660               AAGAATATCAAGTGTGATGTGACAGCCCAGGCCTTCCTGGACGAGACGGCCGTCATGACG720               AAGATGCAACACGAGAACCTGGTGCGTCTCCTGGGCGTGATCCTGCACCAGGGGCTGTAC780               ATTGTCATGGAGCACGTGAGCAAGGGCAACCTGGTGAACTTTCTGCGGACCCGGGGTCGA840               GCCCTCGTGAACACCGCTCAGCTCCTGCAGTTTTCTCTGCACGTGGCCGAGGGCATGGAG900               TACCTGGAGAGCAAGAAGCTTGTGCACCGCGACCTGGCCGCCCGCAACATCCTGGTCTCA960               GAGGACCTGGTGGCCAAGGTCAGCGACTTTGGCCTGGCCAAAGCCGAGCGGAAGGGGCTA1020              GACTCAAGCCGGCTGCCCGTCAAGTGGACGGCGCCCGAGGCTCTCAAACACGGGAAGTTC1080              ACCAGCAAGTCGGATGTCTGGAGTTTTGGGGTGCTGCTCTGGGAGGTCTTCTCATATGGA1140              CGGGCTCCGTACCCTAAAATGTCACTGAAAGAGGTGTCGGAGGCCGTGGAGAAGGGGTAC1200              CGCATGGAACCCCCCGAGGGCTGTCCAGGCCCCGTGCACGTCCTCATGAGCAGCTGCTGG1260              GAGGCAGAGCCCGCCCGCCGGCCACCCTTCCGCAAACTGGCCGAGAAGCTGGCCCGGGAG1320              CTACGCAGTGCAGGTGCCCCAGCCTCCGTCTCAGGGCAGGACGCCGACGGCTCCACCTCG1380              CCCCGAAGCCAGGAGCCC1398                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1521 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ATGGCGGGGCGAGGCTCTCTGGTTTCCTGGCGGGCATTTCACGGCTGTGATTCTGCTGAG60                GAACTTCCCCGGGTGAGCCCCCGCTTCCTCCGAGCCTGGCACCCCCCTCCCGTCTCAGCC120               AGGATGCCAACGAGGCGCTGGGCCCCGGGCACCCAGTGTATCACCAAATGCGAGCACACC180               CGCCCCAAGCCAGGGGAGCTGGCCTTCCGCAAGGGCGACGTGGTCACCATCCTGGAGGCC240               TGCGAGAACAAGAGCTGGTACCGCGTCAAGCACCACACCAGTGGACAGGAGGGGCTGCTG300               GCAGCTGGGGCGCTGCGGGAGCGGGAGGCCCTCTCCGCAGACCCCAAGCTCAGCCTCATG360               CCGTGGTTCCACGGGAAGATCTCGGGCCAGGAGGCTGTCCAGCAGCTGCAGCCTCCCGAG420               GATGGGCTGTTCCTGGTGCGGGAGTCCGCGCGCCACCCCGGCGACTACGTCCTGTGCGTG480               AGCTTTGGCCGCGACGTCATCCACTACCGCGTGCTGGACCGCGACGGCCACCTCACAATC540               GATGAGGCCGTGTTCTTCTGCAACCTCATGGACATGGTGGAGCATTACAGCAAGGACAAG600               GGCGCTATCTGCACCAAGCTGGTGAGACCAAAGCGGAAACACGGGACCAAGTCGGCCGAG660               GAGGAGCTGGCCAGGGCGGGCTGGTTACTGAACCTGCAGCATTTGACATTGGGAGCACAG720               ATCGGAGAGGGAGAGTTTGGAGCTGTCCTGCAGGGTGAGTACCTGGGGCAAAAGGTGGCC780               GTGAAGAATATCAAGTGTGATGTGACAGCCCAGGCCTTCCTGGACGAGACGGCCGTCATG840               ACGAAGATGCAACACGAGAACCTGGTGCGTCTCCTGGGCGTGATCCTGCACCAGGGGCTG900               TACATTGTCATGGAGCACGTGAGCAAGGGCAACCTGGTGAACTTTCTGCGGACCCGGGGT960               CGAGCCCTCGTGAACACCGCTCAGCTCCTGCAGTTTTCTCTGCACGTGGCCGAGGGCATG1020              GAGTACCTGGAGAGCAAGAAGCTTGTGCACCGCGACCTGGCCGCCCGCAACATCCTGGTC1080              TCAGAGGACCTGGTGGCCAAGGTCAGCGACTTTGGCCTGGCCAAAGCCGAGCGGAAGGGG1140              CTAGACTCAAGCCGGCTGCCCGTCAAGTGGACGGCGCCCGAGGCTCTCAAACACGGGAAG1200              TTCACCAGCAAGTCGGATGTCTGGAGTTTTGGGGTGCTGCTCTGGGAGGTCTTCTCATAT1260              GGACGGGCTCCGTACCCTAAAATGTCACTGAAAGAGGTGTCGGAGGCCGTGGAGAAGGGG1320              TACCGCATGGAACCCCCCGAGGGCTGTCCAGGCCCCGTGCACGTCCTCATGAGCAGCTGC1380              TGGGAGGCAGAGCCCGCCCGCCGGCCACCCTTCCGCAAACTGGCCGAGAAGCTGGCCCGG1440              GAGCTACGCAGTGCAGGTGCCCCAGCCTCCGTCTCAGGGCAGGACGCCGACGGCTCCACC1500              TCGCCCCGAAGCCAGGAGCCC1521                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1942 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: human                                                           (B) STRAIN: UT-7                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CGGAGGCCCTCCTGGGGGCGGGCGCGGGGCGCGGCTCGGGGGCGCCCCCTGAGCAGAAAA60                CAGGAAGAACCAGGCTCGGTCCAGTGGCACCCAGCTCCCTACCTCCTGTGCCAGCCGACT120               GGCCTGTGGCAGGCCATTCCCAGCGTCCCCGACTGTGACCACTTGCTCAGTGTGCCTCTC180               ACCTGCCTCAGTTTCCCTCTGGGGGCGATGGCGGGGCGAGGCTCTCTGGTTTCCTGGCGG240               GCATTTCACGGCTGTGATTCTGCTGAGGAACTTCCCCGGGTGAGCCCCCGCTTCCTCCGA300               GCCTGGCACCCCCCTCCCGTCTCAGCCAGGATGCCAACGAGGCGCTGGGCCCCGGGCACC360               CAGTGTATCACCAAATGCGAGCACACCCGCCCCAAGCCAGGGGAGCTGGCCTTCCGCAAG420               GGCGACGTGGTCACCATCCTGGAGGCCTGCGAGAACAAGAGCTGGTACCGCGTCAAGCAC480               CACACCAGTGGACAGGAGGGGCTGCTGGCAGCTGGGGCGCTGCGGGAGCGGGAGGCCCTC540               TCCGCAGACCCCAAGCTCAGCCTCATGCCGTGGTTCCACGGGAAGATCTCGGGCCAGGAG600               GCTGTCCAGCAGCTGCAGCCTCCCGAGGATGGGCTGTTCCTGGTGCGGGAGTCCGCGCGC660               CACCCCGGCGACTACGTCCTGTGCGTGAGCTTTGGCCGCGACGTCATCCACTACCGCGTG720               CTGCACCGCGACGGCCACCTCACAATCGATGAGGCCGTGTTCTTCTGCAACCTCATGGAC780               ATGGTGGAGCATTACAGCAAGGACAAGGGCGCTATCTGCACCAAGCTGGTGAGACCAAAG840               CGGAAACACGGGACCAAGTCGGCCGAGGAGGAGCTGGCCAGGGCGGGCTGGTTACTGAAC900               CTGCAGCATTTGACATTGGGAGCACAGATCGGAGAGGGAGAGTTTGGAGCTGTCCTGCAG960               GGTGAGTACCTGGGGCAAAAGGTGGCCGTGAAGAATATCAAGTGTGATGTGACAGCCCAG1020              GCCTTCCTGGACGAGACGGCCGTCATGACGAAGATGCAACACGAGAACCTGGTGCGTCTC1080              CTGGGCGTGATCCTGCACCAGGGGCTGTACATTGTCATGGAGCACGTGAGCAAGGGCAAC1140              CTGGTGAACTTTCTGCGGACCCGGGGTCGAGCCCTCGTGAACACCGCTCAGCTCCTGCAG1200              TTTTCTCTGCACGTGGCCGAGGGCATGGAGTACCTGGAGAGCAAGAAGCTTGTGCACCGC1260              GACCTGGCCGCCCGCAACATCCTGGTCTCAGAGGACCTGGTGGCCAAGGTCAGCGACTTT1320              GGCCTGGCCAAAGCCGAGCGGAAGGGGCTAGACTCAAGCCGGCTGCCCGTCAAGTGGACG1380              GCGCCCGAGGCTCTCAAACACGGGAAGTTCACCAGCAAGTCGGATGTCTGGAGTTTTGGG1440              GTGCTGCTCTGGGAGGTCTTCTCATATGGACGGGCTCCGTACCCTAAAATGTCACTGAAA1500              GAGGTGTCGGAGGCCGTGGAGAAGGGGTACCGCATGGAACCCCCCGAGGGCTGTCCAGGC1560              CCCGTGCACGTCCTCATGAGCAGCTGCTGGGAGGCAGAGCCCGCCCGCCGGCCACCCTTC1620              CGCAAACTGGCCGAGAAGCTGGCCCGGGAGCTACGCAGTGCAGGTGCCCCAGCCTCCGTC1680              TCAGGGCAGGACGCCGACGGCTCCACCTCGCCCCGAAGCCAGGAGCCCTGACCCCACCCG1740              GTGGCCCTTGGCCCCAGAGGACCGAGAGAGTGGAGAGTGCGGCGTGGGGGCACTGACCAG1800              GCCCAAGGAGGGTCCAGGCGGGCAAGTCATCCTCCTGGTGCCCACAGCAGGGGCTGGCCC1860              ACGTAGGGGGCTCTGGGCGGCCCGTGGACACCCCAGACCTGCGAAGGATGATCGCCCGAT1920              AAAGACGGATTCTAAGGACTCT1942                                                    __________________________________________________________________________

I claim:
 1. An isolated polypeptide having tyrosine kinase activity,comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs. 1, 2, 3, 4 and
 5. 2. An isolated deoxyribonucleic acidcoding for a polypeptide having tyrosine kinase activity, saidpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1, 2, 3, 4 and
 5. 3. A replicable recombinantDNA molecule comprising a replicable expression vector and, operablyinserted in said vector, a deoxyribonucleic acid coding for apolypeptide having tyrosine kinase activity, said polypeptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs.1, 2, 3, 4 and
 5. 4. A microorganism or animal cell transformed with areplicable recombinant DNA molecule comprising a replicable expressionvector and, operably inserted in said vector, a deoxyribonucleic acidcoding for a polypeptide having tyrosine kinase activity, saidpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1, 2, 3, 4 and
 5. 5. A method of screening forchemical substances having the capability to inhibit or activatetyrosine kinase activity, which comprises:contacting a sample materialwith a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs. 1, 2, 3, 4 and 5 to detect a chemicalsubstance having the capability to inhibit or activate the tyrosinekinase activity of at least said polypeptide, wherein said capability ofsaid chemical substance is utilized as a criterion for the detection;and isolating said detected chemical substance from the sample material.